Long-term culture of hepatic oval cells in rats in vitro

doi:10.3969/j.issn.2095-4344.2014.29.013

Abstract

Abstract:

BACKGROUND: Hepatic oval cells are recognized as stem/progenitor cells currently, however, long-term culture of hepatic oval cells can inevitably result in the loss of cell activity.

OBJECTIVE: To explore a long-term culture method of hepatic oval cells in vitro.

METHODS: Partially hepatectomized rat model was established by using 2AAF/PH. The regenerated liver was digested by collagenase, and hepatic oval cells were isolated and purified by density gradient centrifugation and identified by immunocytochemistry. Hepatic oval cells were cultured in a medium containing epidermal growth factor and leukaemia inhibitory factor. Then epidermal growth factor and leukaemia inhibitory factor were removed after several months, and its ability of maintaining stem/progenitor cell activities was determined based on morphology and molecular markers.

RESULTS AND CONCLUSION: Both hepatocyte marker ALB and biliary epithelial cell marker CK-19 were found after hepatic oval cells were cultured in a medium containing epidermal growth factor and leukaemia inhibitory factor for 4 months. While in the absence of epidermal growth factor and leukaemia inhibitory factor, the

expression of fetal liver marker AFP was decreased quickly. These results indicated that hepatic oval cells could expand in a medium containing epidermal growth factor and leukaemia inhibitory factor and maintain stem/progenitor cell activities for a long time.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: hepatocytes, epidermal growth factor, leukemia inhibitory factor, cells, cultured

CLC Number:

R318

Yin Shou-xin, Zhou Zhen, Ma Mei-xue, Yang Jing-bo, Yang Dong-ye. Long-term culture of hepatic oval cells in rats in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(29): 4663-4668.

Figures/Tables 2

References

[1] Poon D, Anderson BO, Chen LT,et al. Management of hepatocellular carcinoma in Asia: consensus statement from the Asian Oncology Summit 2009. Lancet Oncol.2009;10(11): 1111-1118.
[2] Sell S. Cellular origin of hepatocellular carcinomas. Semin Cell Dev Biol. 2002;13(6):419-424.
[3] Oertel M, Shafritz DA.Stem cells, cell transplantation and liver repopulation.Biochim Biophys Acta. 2008;1782(2):61-74.
[4] Fujio K, Evarts RP, Hu Z,et al. Expression of stem cell factor and its receptor, c-kit, during liver regeneration from putative stem cells in adult rat.Lab Invest. 1994;70(4):511-516.
[5] Petersen BE, Goff JP, Greenberger JS,et al. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998;27(2):433-445.
[6] Shupe TD, Piscaglia AC, Oh SH,et al. Isolation and characterization of hepatic stem cells, or "oval cells," from rat livers. Methods Mol Biol. 2009;482:387-405.
[7] Koike H, Taniguchi H.Characteristics of hepatic stem/progenitor cells in the fetal and adult liver.J Hepatobiliary Pancreat Sci. 2012;19(6):587-593.
[8] Tong CM, Ma S, Guan XY.Biology of hepatic cancer stem cells.J Gastroenterol Hepatol. 2011;26(8):1229-1237.
[9] Nakatsura T, Yoshitake Y, Senju S,et al.Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker.Biochem Biophys Res Commun. 2003;306(1):16-25.
[10] He ZP, Tan WQ, Tang YF,et al. Activation, isolation, identification and in vitro proliferation of oval cells from adult rat livers.Cell Prolif. 2004;37(2):177-187.
[11] Hu Z, Evarts RP, Fujio K,et al.Expression of hepatocyte growth factor and c-met genes during hepatic differentiation and liver development in the rat.Am J Pathol. 1993;142(6): 1823-1830.
[12] Dezso K, Jelnes P, László V,et al.Thy-1 is expressed in hepatic myofibroblasts and not oval cells in stem cell-mediated liver regeneration.Am J Pathol. 2007;171(5): 1529-1537.
[13] Apte U, Thompson MD, Cui S,et al.Wnt/beta-catenin signaling mediates oval cell response in rodents.Hepatology. 2008;47(1):288-295.
[14] Majumdar A, Curley SA, Wu X, et al.Hepatic stem cells and transforming growth factor β in hepatocellular carcinoma.Nat Rev Gastroenterol Hepatol. 2012;9(9):530-538.
[15] Tsuchiya A, Heike T, Fujino H, et al.Long-term extensive expansion of mouse hepatic stem/progenitor cells in a novel serum-free culture system.Gastroenterology. 2005;128(7): 2089-2104.
[16] Sirica AE, Mathis GA, Sano N,et al. Isolation, culture, and transplantation of intrahepatic biliary epithelial cells and oval cells.Pathobiology. 1990;58(1):44-64. .
[17] Monga SP, Tang Y, Candotti F,et al.Expansion of hepatic and hematopoietic stem cells utilizing mouse embryonic liver explants.Cell Transplant. 2001;10(1):81-89.
[18] Kamiya A, Kinoshita T, Miyajima A.Oncostatin M and hepatocyte growth factor induce hepatic maturation via distinct signaling pathways.FEBS Lett. 2001;492(1-2):90-94.
[19] Riehle KJ, Dan YY, Campbell JS,et al.New concepts in liver regeneration.J Gastroenterol Hepatol. 2011;26 Suppl 1: 203-212.
[20] Theise ND, Saxena R, Portmann BC,et al.The canals of Hering and hepatic stem cells in humans.Hepatology. 1999; 30(6):1425-1433.
[21] Steinberg P, Steinbrecher R, Schrenk D,et al. Drug-metabolizing enzyme activities in freshly isolated oval cells and in an established oval cell line from carcinogen-fed rats.Cell Biol Toxicol. 1994;10(1):59-65.
[22] Steinberg P, Weisse G, Eigenbrodt E,et al.Expression of L- and M2-pyruvate kinases in proliferating oval cells and cholangiocellular lesions developing in the livers of rats fed a methyl-deficient diet.Carcinogenesis. 1994;15(1):125-127.
[23] Lowes KN, Brennan BA, Yeoh GC,et al.Oval cell numbers in human chronic liver diseases are directly related to disease severity.Am J Pathol. 1999;154(2):537-541.
[24] Fotiadu A, Tzioufa V, Vrettou E,et al. Progenitor cell activation in chronic viralhepatitis.Liver Int. 2004;24(3):268-274.
[25] Huang T, Chesnokov V, Yokoyama KK, et al.Expression of the Hoxa-13 gene correlates to hepatitis B and C virus associated HCC.Biochem Biophys Res Commun. 2001;281(4):1041- 1044.
[26] Steinberg P, Steinbrecher R, Radaeva S,et al. Oval cell lines OC/CDE 6 and OC/CDE 22 give rise to cholangio-cellular and undifferentiated carcinomas after transformation.Lab Invest. 1994;71(5):700-709.
[27] Zhang F, Chen XP, Zhang W,et al.Combined hepatocellular cholangiocarcinoma originating from hepatic progenitor cells: immunohistochemical and double-fluorescence immunostaining evidence.Histopathology. 2008;52(2): 224-232.
[28] Hooth MJ, Coleman WB, Presnell SC,et al. Spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells.Am J Pathol. 1998;153(6):1913-1921.
[29] 刘君,薛玲,张萌,等.肝细胞生长因子和表皮生长因子联合体外诱导大鼠肝卵圆细胞分化的研究[J].中华病理学杂志,2007,36(11): 756-759.
[30] Amit M, Carpenter MK, Inokuma MS,et al.Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.Dev Biol. 2000;227(2):271-278.
[31] Suzuki A, Iwama A, Miyashita H,et al.Role for growth factors and extracellular matrix in controlling differentiation of prospectively isolated hepatic stem cells.Development. 2003; 130(11):2513-2524.
[32] Masson NM, Currie IS, Terrace JD, et al. Hepatic progenitor cells in human fetal liver express the oval cell marker Thy-1.Am J Physiol Gastrointest Liver Physiol. 2006;291(1): G45-54.